Rationale: Positive outward remodeling of pre-existing collateral arteries into functional conductance arteries, arteriogenesis, is a major endogenous rescue mechanism to prevent cardiovascular ischemia. Collateral arterial growth is accompanied by expression of kinin precursor. However, the role of kinin signaling via the kinin receptors (B1R and B2R) in arteriogenesis is unclear.Objective: The purpose of this study was to elucidate the functional role and mechanism of bradykinin receptor signaling in arteriogenesis. Key Words: bone marrow transplantation Ⅲ bradykinin receptors Ⅲ collateral growth Ⅲ leukocytes A rteriogenesis is the process that involves the flow-induced outward remodeling of preexisting collateral arterial pathways into functional conductance arteries (biological bypass). As a result of the arteriogenesis process, blood perfusion to the compromised region is restored; 1 therefore, it is regarded as a clinically highly relevant target. It is established that arteriogenesis is triggered by changes in local hemodynamic conditions and subsequent activation of inflammatory pathways. We previously showed that expression of kininogen, a precursor of the vasoactive kinin peptides, was selectively expressed in growing collaterals of the rat brain. 2 Here we investigated the role of kinin signaling in bradykinin receptor-deficient mice for collateral growth and evaluated whether stimulation with bradykinin receptor antagonists/agonists may modulate arteriogenesis in mice and rats. Our data suggest that the kinin-receptor signaling pathway may act as a molecular link between changes in hemodynamic forces (artery occlusion) and the activation of inflammatory pathways, including attraction of bone marrow Original Methods and Results:

BackgroundIn the presence of a coronary occlusion, pre-existing small collateral vessels (arterioles) develop into much larger arteries (biological bypasses) that have the potential to allow a certain level of perfusion distal to the blockage. Termed arteriogenesis, this phenomenon proceeds via a complex combination of events, with nitric oxide (NO) playing an essential role. The aim of this study was to investigate the effects of supplemental administration of NO donors, i.e., short-acting nitroglycerin (NTG) or slow-release pelleted isosorbide dinitrate (ISDN), on collateral development in a repetitive coronary artery occlusion model in rats.MethodsCoronary collateral growth was induced via a repetitive occlusion protocol (ROP) of the left anterior descending coronary artery (LAD) in rats. The primary endpoints were the histological evaluation of rat heart infarct size and ST-segment elevation (ECG-analysis) upon final permanent occlusion of the LAD (experimentally induced myocardial infarction). The effects of NTG or ISDN were also evaluated by administration during 5 days of ROP. We additionally investigated whether concomitant application of NTG can compensate for the anti-arteriogenic effect of acetylsalicylic acid (ASA).ResultsAfter 5 days of ROP, the mean infarct size and degree of ST-elevation were only slightly lower than those of the SHAM group; however, after 10 days of the protocol, the ROP group displayed significantly less severe infarct damage, indicating enhanced arteriogenesis. Intermittent NTG application greatly decreased the ST-elevation and infarct size. The ISDN also had a positive effect on arteriogenesis, but not to the same extent as the NTG. Administration of ASA increased the infarct severity; however, concomitant dosing with NTG somewhat attenuated this effect.ConclusionIntermittent treatment with the short-acting NTG decreased the size of an experimentally induced myocardial infarct by promoting coronary collateral development. These new insights are of great relevance for future clinical strategies for the treatment of occlusive vascular diseases.

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ObjectiveWe investigated the impact of cardioprotective drugs on ST-elevation, arrhythmias and infarct size in a rat model of repetitive coronary artery occlusion.MethodsSeventy Sprague-Dawley rats were randomised to two control and five treatment groups. Placebo was either implantation of a pneumatic occluder onto the left anterior descending coronary artery (LAD) without starting repetitive occlusion (SHAM) or subsequent RO of the LAD over 10 days without medication (ROP). Treatment groups underwent RO and additionally received nitroglycerin (NTG), metoprolol, verapamil (VER), ranolazine (RAN) or candesartan (CAN). Two weeks after the intervention, rats underwent a single, sustained LAD occlusion followed by reperfusion. To evaluate differences in cardiac resistance against myocardial ischaemia and reperfusion injury, cardiac surrogate parameters including maximal ST-elevation, arrhythmias and infarct size were assessed.ResultsCompared with sham, RO alone and RO plus nitroglycerin were associated with significantly lower maximal ST-elevation and percentage of infarcted myocardium (SHAM 0.12 mV, ROP 0.06 mV (p=0.004), NTG 0.05 mV (p=0.005); SHAM 16.2%, ROP 6.6% (p=0.008), NTG 5.9% (p=0.006). Compared with RO alone, RO plus RAN was accompanied by increased ST-elevation (0.13 mV, p=0.018) and RO plusVER or CAN by more infarcted myocardium (14.2%, p=0.004% and 15.5%, p=0.003, respectively). Rats treated with VER, RAN or CAN tended to severe arrhythmias more frequently than those of the control groups.ConclusionsRO led to an increased myocardial resistance against ischaemia and reperfusion injury. Concomitant administration of nitroglycerin did not affect the efficacy of RO. Cardiovascular channel or receptor blockers reduced the efficacy of RO.

Collateral growth, arteriogenesis, represents a proliferative mechanism involving endothelial cells, smooth muscle cells, and monocytes/macrophages. Here we investigated the role of Density-Enhanced Phosphatase-1 (DEP-1) in arteriogenesis in vivo, a protein-tyrosine-phosphatase that has controversially been discussed with regard to vascular cell biology. Wild-type C57BL/6 mice subjected to permanent left common carotid artery occlusion (CCAO) developed a significant diameter increase in distinct arteries of the circle of Willis, especially in the anterior cerebral artery. Analyzing the impact of loss of DEP-1 function, induction of collateralization was quantified after CCAO and hindlimb femoral artery ligation comparing wild-type and DEP-1−/− mice. Both cerebral collateralization assessed by latex perfusion and peripheral vessel growth in the femoral artery determined by microsphere perfusion and micro-CT analysis were not altered in DEP-1−/− compared to wild-type mice. Cerebrovascular reserve capacity, however, was significantly impaired in DEP-1−/− mice. Cerebrovascular transcriptional analysis of proarteriogenic growth factors and receptors showed specifically reduced transcripts of PDGF-B. SiRNA knockdown of DEP-1 in endothelial cells in vitro also resulted in significant PDGF-B downregulation, providing further evidence for DEP-1 in PDGF-B gene regulation. In summary, our data support the notion of DEP-1 as positive functional regulator in vascular cerebral arteriogenesis, involving differential PDGF-B gene expression.

PTPs exhibit negative regulatory function in cerebral collateral growth in rats. Inhibition of pan-PTP/PTP1B increases vessel PDGF-β receptor phosphorylation. PTP1B inhibition enhances arteriogenesis and cerebrovascular reserve capacity.

In order to assess the extent to which the legally prescribed training for the acquisition of animal experimentation expertise provides scientific personnel with the necessary competence and expertise to carry out a correct harm-benefit analysis in the context of animal experimentation applications, we conducted an interactive stress assessment concerning the basic animal experimentation expertise course. First, before the practical part of the course and then, after the practical part, the participants assessed images and video material of healthy and stressed animals. The results were assessed comparatively and showed a significant increase in performance in all categories (p-value < 0.001). In addition, the results were comparatively assessed against those of scientists already experienced in animal experiments and experienced animal caretakers in research and clinics. In all groups, the vast majority of participants were able to recognise stress in laboratory animals. A significant proportion of the participants were also able to rate the level of stress correctly according to three degrees of severity: mild, moderate and severe. Nevertheless, a small number of participants were unable to distinguish between healthy and stressed animals and thus, the stress in the individual groups was assigned very differently from the different degrees of severity. The results of this study illustrate, on the one hand, the high significance that training must have in order to acquire the expertise, and, on the other hand, how strongly the assessment of stress is influenced by subjectivity.

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